Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.
The synthesis and biochemical properties of oligonucleotides containing phosphorus-modified phosphonoacetate and thio-phosphonoacetate deoxyribonucleotides have been described in scientific journals and patent literature (see Dellinger et al., J. Am. Chem. Soc. 2003, 125(4), 940-950; Sheehan et al., Nucl. Acids Res. 2003, 31(14), 4109-4118); also see published U.S. patent applications (U.S. 2004/0116687 and U.S. 2002/0058802) and U.S. Pat. No. 6,693,187.
DNA or RNA containing oligonucleotides comprising alkylphosphonate internucleoside linkage backbone have been disclosed (see U.S. Pat. Nos. 5,264,423 and 5,286,717).
The synthesis of oligodeoxyribonucleotides containing a methyl phosphonate locked nucleic acid (LNA) thymine monomer has been described. The Tm values of the duplexes with their DNA or RNA complements have also been reported (see Lauritsen et al., Bioorg. Med. Chem. Lett. 2003, 13(2), 253-256).
Oligomeric compounds have been prepared using Click chemistry wherein alkynyl phosphonate internucleoside linkages on an oligomeric compound attached to a solid support are converted into the 1,2,3-triazolylphosphonate internucleoside linkages and then cleaved from the solid support (Krishna et al., J. Am. Chem. Soc. 2012, 134(28), 11618-11631).
Targeting disease-causing gene sequences was first suggested more than thirty years ago (Belikova et al., Tet. Lett. 1967, 8(37), 3557-3562), and antisense activity was demonstrated in cell culture more than a decade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A. 1978, 75(1), 280-284). One advantage of antisense technology in the treatment of a disease or condition that stems from a disease-causing gene is that it is a direct genetic approach that has the ability to modulate (increase or decrease) the expression of specific disease-causing genes. Another advantage is that validation of a therapeutic target using antisense compounds results in direct and immediate discovery of the drug candidate; the antisense compound is the potential therapeutic agent.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and modulates gene expression activities or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi generally refers to antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of targeted endogenous mRNA levels. An additional example of modulation of RNA target function by an occupancy-based mechanism is modulation of microRNA function. MicroRNAs are small non-coding RNAs that regulate the expression of protein-coding RNAs. The binding of an antisense compound to a microRNA prevents that microRNA from binding to its messenger RNA targets, and thus interferes with the function of the microRNA. Regardless of the specific mechanism, this sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of malignancies and other diseases.
Antisense technology is an effective means for reducing the expression of one or more specific gene products and can therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications. Chemically modified nucleosides are routinely used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA. In 1998, the antisense compound, Vitravene® (fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.) was the first antisense drug to achieve marketing clearance from the U.S. Food and Drug Administration (FDA), and is currently a treatment of cytomegalovirus (CMV)-induced retinitis in AIDS patients.
New chemical modifications have improved the potency and efficacy of antisense compounds, uncovering the potential for oral delivery as well as enhancing subcutaneous administration, decreasing potential for side effects, and leading to improvements in patient convenience. Chemical modifications increasing potency of antisense compounds allow administration of lower doses, which reduces the potential for toxicity, as well as decreasing overall cost of therapy. Modifications increasing the resistance to degradation result in slower clearance from the body, allowing for less frequent dosing. Different types of chemical modifications can be combined in one compound to further optimize the compound's efficacy.
The synthesis of 5′-substituted DNA and RNA derivatives and their incorporation into oligomeric compounds has been reported in the literature (Saha et al., J. Org. Chem. 1995, 60, 788-789; Wang et al., Bioorg. Med. Chem. Lett. 1999, 9(6), 885-890; and Mikhailov et al., Nucleosides Nucleotides 1991, 10(1-3), 339-343; Beigelman et al., Nucleosides Nucleotides 1995, 14(3-5), 901-905; and Eppacher et al., Helv. Chim. Acta. 2004, 87, 3004-3020). The 5′-substituted monomers have also been made as the monophosphate with modified bases (Wang et al., Nucleosides Nucleotides Nucleic Acids 2004, 23 (1 & 2), 317-337).
A genus of modified nucleosides including optional modification at a plurality of positions including the 5′-position and the 2′-position of the sugar ring and oligomeric compounds incorporating these modified nucleosides therein has been reported (see International Application Number: PCT/U.S.94/02993, Published on Oct. 13, 1994 as WO 94/22890).
The synthesis of 5′-CH2—R substituted 2′-O-protected nucleosides and their incorporation into oligomers has been previously reported (see Wu et al., Helv. Chim. Acta. 2000, 83, 1127-1143 and Wu et al., Bioconjug. Chem. 1999, 10, 921-924).
Amide linked nucleoside dimers have been prepared for incorporation into oligonucleotides wherein the 3′ linked nucleoside in the dimer (5′ to 3′) comprises a 2′-OCH3 and a 5′-(S)—CH3 (De Mesmaeker et al., Synlett 1997, 11, 1287-1290).
A genus of 2′-substituted 5′-CH2—R (or O) modified nucleosides and a discussion of incorporating them into oligonucleotides has been previously reported (see International Application Number: PCT/U.S.92/01020, published on Feb. 7, 1992 as WO 92/13869).
The synthesis of modified 5′-methylene phosphonate monomers having 2′-substitution and their use to make modified antiviral dimers has been previously reported (see U.S. patent application Ser. No. 10/418,662, published on Apr. 6, 2006 as U.S. 2006/0074035).
Various analogs of 5′-alkynylphosphonate ribonucleosides have been prepared and reported in the literature (see Meurillon et al., Tetrahedron 2009, 65, 6039-6046; Meurillon et al., Nucleic Acids Symp. Ser. 2008, 52(1), 565-566; Lera et al., Org. Lett. 2000, 2(24), 3873-3875).
The preparation of 5′-vinylphosphonate DNA and RNA monomers and their use to make dimeric compounds for oligonucleotide synthesis have been described. Their biochemical studies have also been discussed (see Whittaker et al., Tet. Lett. 2008, 49, 6984-6987; Abbas et al., Org. Lett. 2001, 3(21), 3365-3367; Bertram et al., Biochemistry 2002, 41, 7725-7731; Zhao et al., Tet. Lett. 1996, 37(35), 6239-6242 and Jung et al., Bioorg. Med. Chem. 2000, 8, 2501-2509).
Various BNA's have been prepared and reported in the patent literature as well as in scientific literature, see for example: Singh et al., Chem. Commun. 1998, 4, 455-456; Koshkin et al., Tetrahedron 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett. 1998, 8, 2219-2222; Wengel et al., PCT International Application WO 98-DK393 19980914; Singh et al., J. Org. Chem. 1998, 63, 10035-10039, the text of each is incorporated by reference herein, in their entirety. Examples of issued U.S. patents and published applications include for example: U.S. Pat. Nos. 7,053,207, 6,770,748, 6,268,490 and 6,794,499 and published U.S. applications 2004/0219565, 2004/0014959, 2003/0207841, 2004/0192918, 2003/0224377, 2004/0143114 and 2003/0082807; the text of each is incorporated by reference herein, in their entirety.
The synthesis of various cyclohexitol nucleoside analogs (tetrahydropyran nucleoside analogs) has been reported in the literature, see for example: Verheggen et al., J. Med. Chem. 1995, 38, 826-835; Altmann et al., Chimia 1996, 50, 168-176; Herdewijn et al., Bioorg. Med. Chem. Lett. 1996, 6(13), 1457-1460; Verheggen et al., Nucleosides Nucleotides 1996, 15(1-3), 325-335; Ostrowski et al., J. Med. Chem. 1998, 41, 4343-4353; Allart et al., Tetrahedron. 1999, 55, 6527-6546; Wouters et al., Bioorg. Med. Chem. Lett. 1999, 9, 1563-1566; Brown et al., Drug Dev. Res. 2000, 49, 253-259; published PCT application: WO 93/25565; WO 02/18406; and WO 05/049582; U.S. Pat. Nos. 5,314,893; 5,607,922; and 6,455,507. Various cyclohexitol nucleoside analogs (tetrahydropyran nucleoside analogs) have been described as monomers and have also been incorporated into oligomeric compounds (see for example: Published PCT application, WO 93/25565, published Dec. 23, 1993; Augustyns et al., Nucleic Acids Res. 1993, 21(20), 4670-4676; Verheggen et al., J. Med. Chem., 1993, 36, 2033-2040; Van Aerschol et al., Angew. Chem. Int. Ed. Engl., 1995, 34(12), 1338-1339; Anderson et al., Tetrahedron Lett. 1996, 37(45), 8147-8150; Herdewijn et al., Liebigs Ann. 1996, 1337-1348; De Bouvere et al., Liebigs Ann./Recueil 1997, 1453-1461; 1513-1520; Hendrix et al., Chem. Eur. J. 1997, 3(1), 110-120; Hendrix et al., Chem. Eur. J. 1997, 3(9), 1513-1520; Hossain et al, J. Org. Chem. 1998, 63, 1574-1582; Allart et al., Chem. Eur. J. 1999, 5(8), 2424-2431; Boudou et al., Nucleic Acids Res. 1999, 27(6), 1450-1456; Kozlov et al., J. Am. Chem. Soc. 1999, 121, 1108-1109; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 2653-2656; Kozlov et al., J. Am. Chem. Soc., 1999, 121, 5856-5859; Pochet et al., Nucleosides & Nucleotides, 1999, 18 (4&5), 1015-1017; Vastmans et al., Collection Symposium Series, 1999, 2, 156-160; Froeyen et al., Helv. Chim. Acta. 2000, 83, 2153-2182; Kozlov et al., Chem. Eur. J., 2000, 6(1), 151-155; Atkins et al., Parmazie, 2000, 55(8), 615-617; Lescrinier et al., Chemistry & Biology, 2000, 7, 719-731; Lescrinier et al., Helv. Chim. Acta. 2000, 83, 1291-1310; Wang et al., J. Am. Chem. 2000, 122, 8595-8602; U.S. Patent Application U.S. 2004/0033967; Published U.S. Patent Application U.S. 2008/0038745; Published and Issued U.S. Pat. No. 7,276,592). DNA analogs have also been reviewed in an article (see: Leumann, Bioorg. Med. Chem. 2002, 10, 841-854) which included a general discussion of cyclohexitol nucleoside analogs (under the name: hexitol nucleic acid family).
Oligomeric compounds having phosphodiester linked hexitol nucleic acids (HNA, or 1,5-anhydrohexitol nucleic acids, 3′-H tetrahydropyran nucleoside analogs) have also been prepared for evaluation in cell assays. The different motifs that have been evaluated are fully modified wherein each monomer is a phosphodiester linked hexitol nucleic acid analog and gapped wherein each monomer in the 3′ and 5′ external regions of the oligomeric compound are each phosphodiester linked hexitol nucleic acid analogs and each monomer in the internal region is a phosphorothioate linked deoxyribonucleoside (see: Kang et al., Nucleic Acids Research, 2004, 32(14), 4411-4419; Vandermeeren et al., 2000, 55, 655-663; Flores et al., Parasitol Res., 1999, 85, 864-866; and Hendrix et al., Chem. Eur. J, 1997, 3(9), 1513-1520).
Oligomeric compounds having phosphodiester linked analogs having the 3′-OH group which are referred to in the art as ANA or D-altritol nucleic acids (3′-OH tetrahydropyran nucleoside analogs) have been prepared and evaluated both structurally and in vitro (Allart et al., Chem. Eur. J. 1999, 5(8), 2424-2431).
Chemically modified siRNA's having incorporated hexitol nucleotides (also referred to in the art as HNA, hexitol nucleic acids and tetrahydropyran nucleoside analogs) have been prepared and tested for silencing capacity (see: Published PCT application, WO 06/047842, published May 11, 2006.
Cyclohexenyl nucleic acids (ceNA) and analogs thereof have been reported in the scientific and patent literature as monomers as well as in oligomeric compounds, see for example: Robeyns et al., J. Am. Chem. Soc. 2008, 130(6), 1979-1984; Horváth et al., Tetrahedron Lett. 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc. 2007, 129(30), 9340-9348; Gu et al., Nucleosides Nucleotides Nucleic Acids 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Res. 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallogr. F Struct. Biol. Commun. 2005, F61(6), 585-586; Gu et al., Tetrahedron 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides 2003, 13(6), 479-489; Wang et al., J. Org. Chem. 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Res. 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem. 2001, 66, 8478-82; Wang et al., Nucleosides Nucleotides Nucleic Acids 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem. 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety.
The synthesis of 5′-phosphonate deoxyribonucleoside monomers and dimers having a 5′-phosphate group and their incorporation into oligomeric compounds have been described. Their physico-chemical properties including thermal stability as well as substrate activity toward certain nucleases have also been discussed (see Nawrot et al., Oligonucleotides 2006, 16(1), 68-82).
Nucleosides having a 6′-phosphonate group have been reported wherein the 5′ or/and 6′-position is unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof) (see Fairhurst et al., Synlett 2001, 4, 467-472; Kappler et al., J. Med. Chem. 1986, 29, 1030-1038; Kappler et al., J. Med. Chem. 1982, 25, 1179-1184; Vrudhula et al., J. Med. Chem. 1987, 30, 888-894; Hampton et al., J. Med. Chem. 1976, 19, 1371-1377; Geze et al., J. Am. Chem. Soc. 1983, 105(26), 7638-7640 and Hampton et al., J. Am. Chem. Soc. 1973, 95(13), 4404-4414).
The synthesis and biochemical properties of oligonucleotides containing phosphorus-modified phosphonoacetate and thio-phosphonoacetate deoxyribonucleotides have been described in scientific journals and patent literature (see Dellinger et al., Am. Chem. Soc. 2003, 125, 940-950; Sheehan et al., Nucleic Acids Res. 2003, 31(14), 4109-4118); also see published U.S. patent applications (U.S. 2004/0116687 and U.S. 2002/0058802) and U.S. Pat. No. 6,693,187.
DNA or RNA containing oligonucleotides comprising alkylphosphonate internucleoside linkage backbone have been disclosed (see U.S. Pat. Nos. 5,264,423 and 5,286,717).
The synthesis of oligodeoxyribonucleotides containing a methyl phosphonate locked nucleic acid (LNA) thymine monomer has been described. The Tm values of the duplexes with their DNA or RNA complements have also been reported (see Lauritsen et al., Bioorg. Med. Chem. Lett. 2003, 13(2), 253-256).
Oligomeric compounds have been prepared using Click chemistry wherein alkynyl phosphonate internucleoside linkages on an oligomeric compound attached to a solid support are converted into the 1,2,3-triazolylphosphonate internucleoside linkages and then cleaved from the solid support (Krishna et al., J. Am. Chem. Soc. 2012, 134(28), 11618-11631).